Antimicrobial compositions comprising cannabinoids and methods of using the same

ABSTRACT

The invention relates is directed to antimicrobial compositions comprising cannabinoid compounds and methods of utilizing the antimicrobial compositions to inhibit the growth of microorganisms, to treat and/or prevent microbial infections.

FIELD OF THE INVENTION

The invention relates to antimicrobial compositions comprisingcannabinoid compounds and methods of utilizing the antimicrobialcompositions to inhibit the growth of and/or kill microorganisms, totreat and/or prevent microbial infections.

BACKGROUND OF THE INVENTION

The World Health Organization (WHO) has declared that antibioticresistance is one of the biggest threats to global health, foodsecurity, and development today. The United States Centers for DiseaseControl and Prevention (CDC) estimates that at a minimum there are2,049,442 illnesses caused by antibiotic resistance resulting in 23,000deaths annually.

Overuse of antibiotics is detrimental to the healthy intestinalmicrobiota, and can result in dysbiosis and microbial imbalances andovergrowths of pathogenic microbes such as Clostridium difficile (C.difficile). In 2015, the CDC estimated that nearly a half a millionAmericans suffered from C. difficile infections, which were directlyresponsible for 15,000 deaths and attributed to 29,000 total deaths(10).

The US Centers for Disease Control and Prevention (CDC) published areport in 2013 listing the top 18 drug-resistant threats to the UnitedStates. This list is divided into urgent, serious, and concerning threatlevels. Included in the urgent threat list are Clostridium difficile,carbapenem-resistant Enterobacteriaceae, and cephalosporin-resistantNeisseria gonorrhoeae. Included in the serious threat list aremultidrug-resistant Acinetobacter, drug-resistant Campylobacter,fluconazole-resistant Candida, extended spectrum beta-lactamaseproducing enterobacteriaceae, vancomycin-resistant Enterococcus,multidrug-resistant Pseudomonas aeruginosa, drug-resistant non-typhoidalSalmonella, drug-resistant Salmonella typhi, drug-resistant Shigella,methicillin-resistant Staphylococcus aureus, drug-resistantStreptococcus pneumonia, and drug-resistant tuberculosis. Included inthe concerning threat list are vancomycin-resistant Staphylococcusaureus, erythromycin-resistant Streptococcus group A, andclindamycin-resistant Streptococcus group B (9).

In February 2017, the WHO published a list of priority pathogens forresearch and development of new antibiotics. This list is divided intothree main groups of critical, high, and medium priority. Included inthe critical list are carbapenem-resistant Acinetobacter baumannii,Pseudomonas aeruginosa, and Enterobacteriacea. Included in the highpriority list are vancomycin-resistant Enterococcus faecium,methicillin-resistant and vancomycin intermediate and resistantStaphylococcus aureus, clarithromycin-resistant Helicobacter pylori,fluoroquinolone-resistant Campylobacter and Salmonella spp., and 3rdgeneration cephalosporin-resistant and fluoroquinolone-resistantNeisseria gonorrhoeae. Included in the medium priority list arepenicillin-non-susceptible Streptococcus pneumoniae,ampicillin-resistant Haemophilus influenzae, andfluoroquinolone-resistant Shigella spp (15).

With the emergence of antimicrobial-resistance and the urgent threat tohuman health it represents, there is a critical need to develop novelantibiotics that are safe and effective. Van Klingern and Ten Hamobserved that THC and CBD inhibited the growth of Staphlococcus aureusin the 1-5 μg/ml range (13). It has been shown that Cannabis sativaindicus extracts were effective as an antimicrobial agent towardsClostridium perfringens (12). Ali 2012 conducted antimicrobial testingof petroleum and alcohol extracts of cannabis. The extracts demonstratedactivity against MRSA, but were ineffective against gram-negativebacteria and fungi (7). Appendino et al. 2008 published astructure-activity study comparing the structures of differentcannabinoids for their ability to inhibit the growth of bacteriaincluding MRSA (8).

To date, no evaluation of the antimicrobial activity of cannabinoids hasbeen performed towards Clostridium difficile or Enterococcus faecium.

SUMMARY OF THE INVENTION

The present invention provides antimicrobial compositions comprisingcannabinoids and methods of using the same. In certain aspects of thepresent invention, there is provided a composition having antimicrobialactivity comprising a cannabinoid compound or a prodrug that ismetabolized to the cannabinoid compound. In certain embodiments, thecannabinoid compound is cannabidiol, tetrahydrocannabinol, or a prodrugthat is metabolized to cannabidiol or tetrahydrocannabinol, optionally aglycoside of cannabidiol or tetrahydrocannabinol. The composition may beformulated for oral administration or topical administration. In certainembodiments, the composition comprises a further active ingredientselected from other cannabinoid compounds, other antimicrobial agents,anti-inflammatory agents, probiotics and combinations thereof. Theantimicrobial activity may be against gram positive aerobic bacteriaand/or anaerobic bacteria.

In other aspects of the present invention, there is provided a method oftreating an infection comprising applying to a patient in need thereofat the site of infection an effective amount of a topical compositioncomprising an effective amount of a cannabidiol or tetrahydrocannabinolin a pharmaceutically acceptable carrier for topical application. Incertain embodiments, the infection is S. pyogenes infection.

In other aspects of the present invention, there is provided a method oftreating microbial infections of the gastrointestinal tract, comprisingoral administration of an effective amount of a cannabinoid compound. Incertain embodiments, the cannabinoid compound is a glycoside ofcannabidiol or tetrahydrocannabinol. In certain embodiments, theinfection is a C. difficile infection; a methicillin-resistantStaphylococcus aureus infection or an E. faecalis infection.

In other aspects of the present invention, there is provided a method ofinhibiting the growth of and/or killing microorganisms comprisingadministration of an effective amount of a cannabinoid compound. Incertain embodiments, the microorganism is bacteria. In certainembodiments, the cannabinoid compound is cannabidiol ortetrahydrocannabinol.

DESCRIPTION OF THE FIGURES

These and other features of the invention will become more apparent inthe following detailed description in which reference is made to theappended drawings.

FIG. 1A provides the chemical structures of VB102, VB106, VB110 andVB302.

FIG. 1B provides the chemical structures of glycosides of cannabidioland Δ9-THC; wherein each R is independently H, mono, di ortri-glycosidic residue.

FIG. 2A illustrates VB110 and CBD decoupling analysis of extracts fromsmall intestinal contents.

FIG. 2B illustrates VB110 and CBD decoupling analysis of extracts fromlarge intestinal contents.

DESCRIPTION OF THE INVENTION

The invention is based on the discovery that cannabinoid compoundsinhibit the growth of and/or kill certain types of microorganisms.Accordingly, the present invention provides antimicrobial compositionscomprising a cannabinoid compound alone or in combination with otheragents and methods of utilizing the antimicrobial compositions toinhibit the growth of and/or kill microorganisms, to treat and/orprevent microbial infections.

The cannabinoid compounds include any cannabinoid compound which hasantimicrobial activity or prodrugs thereof. A worker skilled in the artwould readily appreciate that antimicrobial agents may have broadspectrum antimicrobial activity (i.e. activity against a wide range ofmicroorganisms including bacteria, yeast, and fungus), may haveantibacterial activity (i.e. activity against bacteria only), or mayhave activity against a selection of specific microorganisms or a singlemicroorganism. For example, the agent may be active againstgram-positive bacteria or gram-negative bacteria, may be active againstaerobic bacteria or anaerobic bacteria or may be active against a subsetthereof, for example, the agent may be active against gram-positiveaerobes or gram-positive anaerobes. In certain embodiments, thecannabinoid compound has antimicrobial activity against gram-positivebacteria. In specific embodiments, the cannabinoid compound hasantimicrobial activity against gram-positive aerobic bacteria. In otherspecific embodiments, the cannabinoid compound has antimicrobialactivity against gram-positive anaerobic bacteria.

Non-limiting examples of gram-positive aerobic bacteria includeStaphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniaand Streptococcus pyogenes. In certain embodiments, the cannabinoidcompound has antimicrobial activity against Staphylococcus aureus. Inspecific embodiments, the cannabinoid compound has antimicrobialactivity against methicillin-sensitive Staphylococcus aureus (MSSA). Inspecific embodiments, the cannabinoid compound has antimicrobialactivity against methicillin-resistant Staphylococcus aureus (MRSA). Incertain embodiments, the cannabinoid compound has antimicrobial activityagainst Enterococcus faecalis. In specific embodiments, the cannabinoidcompound has antimicrobial activity against vanomycin-sensitiveEnterococcus faecalis (VSE). In specific embodiments, the cannabinoidcompound has antimicrobial activity against vanomycin-resistantEnterococcus faecalis (VRE). In certain embodiments, the cannabinoidcompound has antimicrobial activity against Streptococcus pneumonia. Incertain embodiments, the cannabinoid compound has antimicrobial activityagainst Streptococcus pygenes.

Non-limiting examples of anaerobic bacteria include Clostridiumdifficile, Lactobacillus crispatus, Lactobacillus jensenii,Bifidobacterium bifidum, Bifidobacterium longum and Bacteroidesfragilis. In certain embodiments, the cannabinoid compound hasantimicrobial activity against Clostridium difficile. In certainembodiments, the cannabinoid compound has antimicrobial activity againstLactobacillus sp. In certain embodiments, the cannabinoid compound hasantimicrobial activity against Bifidobacterium sp.

Antimicrobial activity may be microcidal (kills the microorganism)and/or biostatic (inhibits the growth of the microorganism).Accordingly, in certain embodiments, the cannabinoid compound exhibitsmicrocidal activity. In certain embodiments, the cannabinoid compoundinhibits the growth of microorganisms. In certain embodiments, thecannabinoid compound has microcidal activity. In certain embodiments,the cannabinoid compound has microcidal activity and also inhibits thegrowth of microorganisms.

The term “cannabinoid compound” as used herein generally refers tocompounds found in cannabis or which act on cannabinoid receptors andalso includes prodrugs thereof. The cannabinoid compounds may beisolated from cannabis or may be synthetic.cannabinoid compounds. Thecannabinoid compounds include, but are not limited to, cannabidiol(CBD), cannabidivarin (CBDV), cannabigerol (CBG), tetrahydrocannabinol(Δ9-THC or THC), cannabinol (CBN), cannabidiolic acid (CBDA),tetrahydrocannabivarin (THCV), phytocannabinoids, endocannabinoids, andprodrugs thereof. In specific embodiments, the cannabinoid compound iscannabidiol. In specific embodiments, the cannabinoid compound istetrahydrocannabinol.

The cannabinoid compounds may be in the form of a prodrug. A workerskilled in the art would readily appreciate that a prodrug is a compoundwhich, upon administration, must undergo a chemical conversion bymetabolic processes before becoming an active pharmacological agent (forexample, have antimicrobial activity). Non-limiting examples of prodrugsinclude glycosides. In certain embodiments, the glycoside is a mono, di,tri or tetra-glycoside of the cannabinoid compound. The glucose residuesof glycosides are commonly acid-hydrolyzed in the stomach or cleaved byglycosidase enzymes in the intestinal tract, including byalpha-glycosidases and beta-glycosidases, which are expressed byintestinal microflora across different regions of the intestine.Accordingly, glycosides are hydrolyzed upon ingestion to release thedesired compound into the intestines or target tissues.

In certain embodiments, the cannabinoid compound is a cannabinoidglycoside prodrug which is capable of persisting in the acidic stomachenvironment upon oral administration and thereby allows for the deliveryof the prodrug into the large intestine, where the cannabinoid aglyconescan be liberated by glycosidases produced by colonic bacteria.

In certain embodiments, the cannabinoid compound is a glycoside ofcannabidiol. In specific embodiments, the glycoside of cannabidiol is amono, di, tri or tetra-glycoside of cannabidiol. In specificembodiments, the glycoside of cannabidiol has a structure set forth inFIG. 1A, wherein each R is independently H, mono, di or tri-glycosidicresidue. In specific embodiments, the glycoside of cannabidiol has astructure selected from VB102, VB106 and VB110 set forth in FIG. 1B.

In certain embodiments, the cannabinoid compound is a glycoside oftetrahydrocannabinol. In specific embodiments, the glycoside oftetrahydrocannabinol is a mono, di, tri or tetra-glycoside oftetrahydrocannabinol. In specific embodiments, the glycoside oftetrahydrocannabinol has a structure as set forth in FIG. 1A, whereineach R is independently H, mono, di or tri-glycosidic residue. Inspecific embodiments, the glycoside of tetrahydrocannabinol has thestructure of VB302 as set forth in FIG. 1B.

The present invention also provides methods of utilizing theantimicrobial compositions. The antimicrobial compositions may beutilized to inhibit the growth of and/or kill microorganisms, and/or totreat and/or prevent microbial infections.

The antimicrobial compositions may be administered by various routesincluding but not limited to topical administration and oraladministration. A person skilled in the art would readily appreciatethat the cannabinoid compound used in the antimicrobial composition maybe dependent on the administration route and microorganism. For example,such a person would readily appreciate that glycoside prodrugs aremetabolized in the gastrointestinal tract for activity and as such mayhave optimal activity in oral drug formulations.

In certain embodiments, there is provided a method of treating a skin orwound infection comprising topically applying to a patient in needthereof at the site of infection an effective amount of a topicalcomposition comprising an effective amount of a cannabinoid compoundwhich has antimicrobial activity, including in a pharmaceuticallyacceptable carrier for topical application. In specific embodiments, thecannabinoid compound is cannabidiol. In specific embodiments, thecannabinoid compound is tetrahydrocannabinol. Appropriatepharmaceutically acceptable carriers are known in the art. In specificembodiments, the topical composition is for treatment of a wound isinfected with methicillin-resistant Staphylococcus aureus orStreptococcus pyogenes.

In certain embodiments, there is provided a method of treating microbialinfections of the gastrointestinal tract, comprising oral administrationof an effective amount of a cannabinoid compound. The cannabinoidcompound may be an active cannabinoid compound or a prodrug thereof. Inspecific embodiments, the cannabinoid compound is cannabidiol. Inspecific embodiments, the cannabinoid compound is a glycoside ofcannabidiol. In specific embodiments, the glycoside of cannabidiol has astructure selected from structures set forth in FIGS. 1A and 1B. Inspecific embodiments, the cannabinoid compound is tetrahydrocannabinolor a glycoside of tetrahydrocannabinol. Optionally, the glycoside oftetrahydrocannabinol has a structure as set forth FIG. 1A or 1B.

In certain embodiments, the microbial infection of the gastrointestinaltract is a Clostridium difficile infection. In certain embodiments, themicrobial infection of the gastrointestinal tract is a Staphylococcusaureus infection, including but not limited to methicillin-resistantStaphylococcus aureus (MRSA) infection. In certain embodiments, themicrobial infection of the gastrointestinal tract is an Enterococcusfaecalis infection, including but not limited to a vancomycin-resistantEnterococcus faecalis infection.

In certain embodiments, the compositions are used in methods of treatingdysbiosis. In specific embodiments, the compositions are used in methodsof treating dysbiosis of the gastrointestinal tract.

The cannabinoid compounds may have activities other than antimicrobialactivities. For example, the compounds may have positive effects on thehealth of the skin or tissue epithelium, including the intestinalepithelium. For example, the compounds may exert anti-inflammatoryeffects.

In certain embodiments, the cannabinoid compound is inhibitory ofharmful pathogens, and also has anti-inflammatory effects. In specificembodiments, the infection is a C. difficile infection and the compoundis cannabidiol in the large intestine is inhibitory of harmful pathogensincluding C. difficile, and it also is anti-inflammatory, where it mayhelp induce or maintain remission of colitis, which is often comorbidwith C. difficile infections.

A person skilled in the art would appreciate that the compositions mayinclude a single cannabinoid compound as the active ingredient or mayfurther comprise other active ingredients including but not limited toother cannabinoid compounds, other antimicrobial agents,anti-inflammatory agents, and/or probiotics.

The following examples illustrate embodiments of the invention butshould not be viewed as limiting the scope of the invention.

EXAMPLES Example 1 Antimicrobial Activity of Cannabidiol and ProdrugsThereof Itemized Summary of Findings:

-   -   CBD inhibits the growth of Clostridium difficile at 32 μg/ml        (IC50)    -   CBD inhibits the growth of methilcillin-resistant Staphylococcus        aureus (MRSA) at 2 μg/ml (IC50)    -   CBD inhibits the growth of vancomycin resistant Enterococcus        faecalis at 2 μg/ml (IC50)    -   CBD inhibits the growth of Streptococcus pneumoniae at 4 μg/ml        (IC50)    -   CBD inhibits the growth of Bacterioides fragilis at 32 μg/ml        (IC50)    -   CBD inhibits the growth of healthy intestinal bacteria including        Lactobacillus crispatus, Lactobacillus jensenii, Bifidobacterium        bifidum, Bifidobacterium longum (2 μg/ml, 64 ug/ml, 2 μg/ml, 32        μg/ml IC50 values respectively).    -   CBD did not have any effect on the gram-negative pathogens        Klebsiella, Pseudomonas, or Acinetobacter.

INTRODUCTION

The primary purpose of this study was to evaluate the in vitrosusceptibility of a variety of clinically important microorganisms tocannabidiol (CBD), an active cannabinoid isolated from cannabis, andother cannabinoid prodrugs (VB104, VB110, and compounds listed in TableA). The in vitro activity of CBD and the cannabinoid prodrugs along withcontrol agents (ciprofloxacin for aerobic bacteria, metronidazole foranaerobic bacteria, and amphotericin B for yeast/fungi) was determinedby broth microdilution minimal inhibitory concentration (MIC) testingconducted in accordance with guidelines from the Clinical and LaboratoryStandards Institute (CLSI; 1, 2).

MATERIALS AND METHODS Test Compounds

The cannabinoid prodrug test agents were stored at −20° C. prior totesting. Prodrugs were supplied as powders. CBD was supplied by CaymanChemical as a 10 mg/mL methanolic solution and was stored at −20° C.after receipt. Prior to testing, using a 614 μL aliquot of the CBDmethanolic solution, the methanol was dried off and the residual CBD wasresuspended in 600 μL DMSO resulting in a 10.24 mg/mL solution. Stocksolutions of comparator compounds were prepared on the day of testingusing solvents recommended by CLSI. Stock solutions of all compoundswere made at 40× the final testing concentration. Information regardingtesting concentrations and drug diluent for the comparator and testagent is detailed in the table below.

Testing Concentration Test Agent Range (μg/ml) Solvent/DiluentCiprofloxacin  64-0.002 Water/Water Metronidazole 32-0.03 DMSO/WaterAmphotericin B 64-0.06 DMSO/DMSO VB104 256-0.002 DMSO/DMSO VB304256-0.002 DMSO/DMSO VB110 256-0.002 DMSO/DMSO CBD 256-0.002 DMSO/DMSO

Test Organisms

The test organisms evaluated in this study consisted of clinicalisolates from the Micromyx Repository and reference isolates from theAmerican Type Culture Collection (ATCC; Manassas, Va.). Upon initialreceipt, the organisms were sub-cultured onto an appropriate agarmedium. Following incubation, colonies were harvested from these platesand cell suspensions prepared and frozen at −80° C. with acryoprotectant. Prior to testing, the isolates were streaked from frozenvials onto Trypticase Soy Agar with 5% sheep blood (BD; Sparks, Md.; LotNo. 107682) for aerobic bacteria, Supplemented Brucella Agar (BD; LotNo.6168880) for anaerobic bacteria, and Sabouraud Dextrose Agar (BD; LotNo. 6265646) for yeast. Aerobic bacteria were incubated at 35° C.overnight, anaerobic bacteria were incubated anaerobically at 35° C. for48 hr, and yeast were incubated at 35° C. for 48 hr. Aspergillusfumigatus was previously streaked on SDA and incubated at 35 oC untilspore formation occurred, followed by harvesting of the spores insterile 0.85% saline and enumerating. Spore preparations were stored at4° C.

Test Medium

Mueller Hinton II broth (MHB II; BD; Lot No. 6258541) was used for MICtesting of aerobic organisms. For Streptococcus isolates, this mediumwas supplemented with 3% laked horse blood (LHB; Cleveland Scientific,Bath, Ohio; Lot No. 343287). MIC testing of anaerobic bacteria wasperformed with Brucella broth (BD; Lot No. 1297468) supplemented withhemin (Sigma, St. Louis, Mo.; Lot No. SLBC4685V), vitamin K1 (Sigma; LotNo. MHBX5269V), and 5% LHB. Yeast and fungi were tested in RPMI-1640from Hyclone Laboratories (Logan, Utah; Lot No. AZC984041B) bufferedwith MOPs from Calbiochem (Billerica, Mass.; Lot No. 2730641).

Broth Microdilution MIC Methodology

MIC values were determined using a broth microdilution proceduredescribed by CLSI (1-5). Automated liquid handlers (Multidrop 384,Labsystems, Helsinki, Finland; Biomek 2000 and Biomek FX, BeckmanCoulter, Fullerton Calif.) were used to conduct serial dilutions andliquid transfers. To prepare the drug mother plates, which would providethe serial drug dilutions for the replicate daughter plates, the wellsof columns 2-12 of standard 96-well microdilution plates (Costar 3795)were filled with 150 μl of the designated diluent for each row of drug.The test articles and comparator compounds (300 μl at 40× the highestconcentration to be tested) were dispensed into the appropriate wells incolumn 1. The Biomek 2000 was then used to make 2-fold serial dilutionsin the mother plates from column 1 through column 11. The wells ofColumn 12 contained no drug and served as the organism growth controlwells for the assay. The daughter plates were loaded with 185 μL perwell of the appropriate test medium for the tested organism using theMultidrop 384. The daughter plates were completed on the Biomek FXinstrument which transferred 5 μL of drug solution from each well of amother plate to the corresponding well of each daughter plate in asingle step. Daughter plates for the testing of anaerobes were allowedto pre-reduce in the Bactron II anaerobe chamber for 2 hr prior toinoculation.

A standardized inoculum of each test organism was prepared per CLSImethods (1-5). The inoculum for each organism was dispensed into sterilereservoirs divided by length (Beckman Coulter), and the Biomek 2000 wasused to inoculate the plates. Daughter plates were placed on the Biomek2000 work surface reversed so that inoculation took place from low tohigh drug concentration. The plates were then inoculated with 10 μL ofthe inoculum resulting in a final concentration of approximately 5×10 5CFU/mL (bacteria), 0.5 to 2.5×10 3 CFU/mL (yeast), and 0.2 to 2.5×10 4CFU/mL (filamentous fungi) per well.

Plates were stacked 3-4 high, covered with a lid on the top plate,placed in plastic bags, and incubated at 35° C. for approximately 16 to20 hr (aerobes), 20 to 24 hr (Streptococcus), and 24 and 48 hr (fungi).Anaerobe plates were stacked, covered with a lid on the top plate,placed in a BD GasPak EZ Anaerobe Container System and incubated at 35°C. for 48 hr. Following incubation, the microplates were removed fromthe incubator and viewed from the bottom using a plate viewer. For eachof the test media and each drug, an un-inoculated solubility controlplate was observed for evidence of drug precipitation. The MIC was readand recorded as the lowest concentration of drug that inhibited visiblegrowth of the organism.

RESULTS AND DISCUSSION

Compound precipitation observed with the uninoculated solubilitycontrols is summarized in the following table.

Compound and lowest concentration (μg/mL) at which precipitation wasobserved Media type VB104 VB304 VB110 CBD MHB II TRP-16 NA none TRP-16MHB II with Blood TRP-128 NA NA TRP-128 Brucella Broth NA NA NA NA RPMITRP-256 NA TRP-32 NA NA = not applicable (no precipitation observed);TRP = trace precipitate; DCP = distinct precipitate

Trace precipitation was noted for other compounds in various testmedium, but in no instance did trace precipitation interfere with thereading of the MIC. The activity of CBD, the cannabinoid prodrugs, andcontrol agents are shown in the tables below. Results for the controlagents (ciprofloxacin, metronidazole, and amphotericin B) were allwithin the established CLSI QC ranges (2, 6) for the respective agentsand quality control organisms.

A pro-drug must typically undergo a chemical conversion by metabolicprocesses before becoming an active pharmacological agent. Accordingly,the cannabinoid prodrugs (prior to chemical conversion to cannabidiol)were inactive towards gram-positive and -negative aerobic bacteria, CBDwas active against gram-positive aerobes but was inactive againstgram-negative aerobes. Ciprofloxacin activity was as expected for abroad spectrum anti-bacterial agent, with resistance apparent for theevaluated MRSA, VRE, and KPC-2 isolates where fluoroquinolone-resistanceis commonly encountered.

Activity of Cannabinoid Prodrugs, Cannabidiol (CBD), and CiprofloxacinAgainst Aerobic Bacteria

MIC (μg/mL) Cipro- Organism Isolate No. VB104 VB304 VB110 CBD floxacinGram- Staphylococcus MMX 100 >256 >256 >256 2 0.5 positive aureus (MSSA)(ATCC 29213) (0.12-0.5)¹ Staphylococcus MMX 757 >256 >256 >256 2 >64aureus (MRSA) Enterococcus MMX 101 >256 >256 >256 2 1 faecalis (VSE)(ATCC 29212) (0.25-2)¹   Enterococcus MMX 848 >256 >256 >256 2 32faecalis (VRE) Streptococcus MMX 1195 >256 >256 >256 4 1 pneumoniae(ATCC 49619) Streptococcus MMX 404 >256 >256 >256 16 0.5 pyogenes Gram-Escherichia MMX 102 >256 >256 >256 >256 0.008 negative coli (ATCC 25922) (0.004-0.015)¹ Klebsiella MMX 4683 >256 >256 >256 >256 >64 pneumoniae(KPC-2) Pseudomonas MMX 1380 >256 >256 >256 >256 4 aeruginosaAcinetobacter MMX 1630 >256 >256 >256 >256 1 baumannii MSSA =methicillin-susceptible S. aureus; MRSA = methicillin-resistant S.aureus; VSE = vancomycin-susceptible enterococci; VRE =vancomycin-resistant enterococci; KPC-2 = Klebsiella pneumoniaecarbapenemase-positive ¹CLSI QC range shown in parenthesis

As shown in the table below, CBD was active with MICs from 2-64 μg/mL.Metronidazole was active against all organisms with the exception of theevaluated Lactobacillus spp., which is consistent with the anticipatedactivity for these isolates.

Activity of Cannabinoid Prodrugs, Cannabidiol (CBD), and MetronidazoleAgainst Anaerobic Bacteria

MIC (μg/mL) Metroni- Organism Isolate No. VB104 VB304 VB110 CBD dazoleClostridium MMX 4381 >256 >256 >256 32 0.12 difficile (ATCC 00057)Clostridium MMX 8330 >256 >256 >256 32 0.12 difficile Clostridium MMX8333 >256 >256 >256 32 0.5 difficile Lactobacillus MMX4147 >256 >256 >256 2 >32 crispatus Lactobacillus MMX4149 >256 >256 >256 64 >32 jensenii Bifidobacterium MMX3965 >256 >256 >256 2 1 bifidum Bifidobacterium MMX 3968 >256 >256 >25632 8 longum Bacteroides MMX 123 >256 >256 >256 32 0.5 fragilis (ATCC5285) (0.25-2)¹ ¹CLSI QC range shown in parenthesis

Neither the cannabinoid prodrugs or CBD were active against theevaluated yeast and fungal isolates though it is important to note thatsome inhibition of growth (50% relative to the growth control) wasapparent with VB304 at 24 hr against C. parapsilosis at the highest testconcentration (256 μg/mL) and for VB110 at 24 and 48 hr against C.parapsilosis at concentrations>16 μg/mL. Amphotericin B had the expectedactivity against both test isolates.

Activity of Cannabinoid Prodrugs, Cannabidiol (CBD), and Amphotericin BAgainst Yeast and Fungi

MIC (μg/mL) at 24, 48 hr Amphoter- Organism Isolate No. VB104 VB304VB110 CBD icin B Candida MMX 2323 >256, >256¹, >256², >256, 0.5(0.5-2)³  parapsilosis (ATCC 22019) >256 >256 >256² >256  1 (0.25-4)³Aspergillus MMX 5280 >256, >256, >256, >256, 0.5, fumigatus (ATCCMYA-3626) >256 >256 >256 >256 2 (0.5-4)³ ¹50% inhibition observed at 256μg/mL relative to the growth control ²50% inhibition observed at ≥16μg/mL relative to the growth control ³CLSI QC ranges shown inparenthesis

In summary, CBD was active against gram-positive aerobic bacteria (S.aureus, E. faecalis, S. pneumoniae, and S. pyogenes) and both obligateand facultative anaerobes (C. difficile, B. fragilis, Bifidobacteriumspp., and Lactobacillus spp.), but not gram-negative aerobic bacteria oryeast and fungi. The cannabinoid prodrugs had no in vitro activityagainst any of the evaluated microorganisms, though some inhibitoryactivity against the tested yeast isolate was noted for VB110.

As shown in the following examples, the cannabinoid prodrugs aremetabolized in the large intestine to release active compound.Accordingly, a person skilled in the art would expect these prodrugs tohave antimicrobial activity following oral administration.

Example 2 Bioavailability Assay

In order to investigate the effectiveness of glycosylation to effectsite-specific drug delivery, VB110 was administered to three mice byoral gavage and the animals sacrificed at 30, 60, and 90 minutes.Eight-week old male Swiss mice were fasted for 12 hours prior toadministration of 120 mg/kg VB110 in 10% Ethanol USP, 10% PropyleneGlycol USP, 0.05% Sodium Deoxycholate USP, 79.95% Saline USP. Followingtermination and tissue harvest, the intestinal contents were thenextracted and analyzed by C18 reverse phase HPLC. As shown in FIG. 2A,the small intestinal contents showed intact VB110, but no decoupled CBD.As shown in FIG. 2B, the large intestinal contents contained both VB110and CBD in the 60 and 90 minute time points. This decoupling of VB110 isconsistent with the large intestinal decoupling seen for sennosidebeta-glycosides and is the result of secreted beta-glycosidases from thelarge intestinal microflora.

Example 3 Analysis of Large Intestine Contents Upon Administration ofCBD and CBD Glycosides

In order to investigate the metabolism and decoupling of CBD-glycosidesin the large intestine, an aqueous solution of a mixture ofCBD-glycosides was administered to a mouse by oral gavage. As a control,a solution of CBD in cremophor, ethanol, and saline was administered toa second mouse. The animals were each sacrificed at 2 hours. Followingtermination and tissue harvest, the intestinal contents were thenextracted and analyzed by C18 reverse phase HPLC. The mice employed inthis example were eight week old male Swiss mice fasted for 12 hoursprior to administration of the solutions.

The resulting extracts were analyzed by LCMS performed using a ShimadzuLC-MS 2010 EV. Electrospray ionization (ESI) was performed in negativemode. The column was a Silia Chrom XDB C18 5 um, 150 A, 4.6×50 mm. Themethod was 12 min 5 to 95 H₂O:ACN. Acetic acid and formic acid were usedas sample additives during analysis, and the injection volume was 20 μl.

Analysis of the large intestinal contents of animals administered amixture of oral CBD-glycosides indicated that both aglycone andglycosides were present, along with hydroxy metabolites of each:

-   [CBD−H], [2CBD−H] and [CBD*2OH+Formic acid−H] MS data: LC/ESI-LRMS.    [M−H]⁻ (C₂₁H₂₉O₂) Calcd: m/z=313. Found: m/z=313. [2M−H]⁻ (C₄₂H₅₉O₄)    Calcd: m/z=627. Found: m/z=627. [M_(*2OH)+Formic acid−H]⁻ (C₂₂H₃₁O₆    ⁻) Calcd: m/z=391. Found: m/z=391.-   [CBDg1−H], [CBDg1+Cl] and [2CBDg1−H] MS data: LC/ESI-LRMS.    [M_(g1)−H]⁻ (C₂₇H₃₉O₇) Calcd: m/z=475. Found: m/z=475. [M_(g1)+Cl]⁻    (C₂₇H₄₀O₇Cl) Calcd: m/z=511. Found: m/z=511. [2M_(g1)−H]⁻    (C₅₄H₇₉O₁₄) Calcd: m/z=951. Found: m/z=951.-   [CBDg2−H] and [CBDg2+Acetic acid−H] MS data: LC/ESI-LRMS.    [M_(g2)−H]⁻ (C₃₃H₄₉O₁₂) Calcd: m/z=637. Found: m/z=637.    [M_(g2)+Acetic acid−H]⁻ (C₃₅H₅₃O₁₄ ⁻) Calcd: m/z=697. Found:    m/z=697.-   [CBDg3−H], [CBDg3*OH−H] and [CBDg3*OH−2H] MS data: LC/ESI-LRMS.    [M_(g3)−H]⁻ (C₃₉H₅₉O₁₇) Calcd: m/z=799. Found: m/z=799.    [M_(g3*OH)−H]⁻ (C₃₉H₅₉O₁₈) Calcd: m/z=815. Found: m/z=815.    [M_(g3*OH)−2H]⁻²(C₃₉H₅₈O₁₈) Calcd: m/z=407. Found: m/z=407.

Analysis of the large intestinal contents of animals administered oralCBD indicated that hydroxy

-   [CBD*2OH+Formic acid−H] and [2CBD*3OH+Acetic acid−H] MS data:    LC/ESI-LRMS. [M_(*2OH)+Formic acid−H]⁻ (C₂₂H₃₁O₆ ⁻) Calcd: m/z=391.    Found: m/z=391. [2M_(*3OH)+Acetic acid−H]⁻ (C₄₄H₆₃O₁₂ ⁻) Calcd:    m/z=783.9. Found: m/z=784.

The plasma and brains from the same animals were also extracted andanalyzed by HPLC for the presence of CBD-glycosides and CBD. CBD wasonly present in the control animal that received CBD aglycone (data notshown). The contents of the small intestines from the same animals werealso extracted and analyzed by HPLC for the presence of CBD-glycosidesand CBD, but no CBD aglycone was present in the small intestines (datanot shown). The presence of the CBD aglycone in the large intestinalcontents indicates the successful delivery of CBD-glycosides, and thesubsequent hydrolysis of the glycosides by beta-glycosidase enzymes onlypresent in the large intestine. The presence of decoupled CBD in thelarge intestine, but not in the small intestine, indicates thatglycoside decoupling only occurs upon transit to the large intestine.The presence of CBD detoxification metabolite CBD-2OH is also consistentwith delivery of CBD and absorption into the intestinal epithelium whereCBD begins to be metabolized. This example illustrates the potential toadminister CBD-glycosides, safely transit the CBD-glycosides through thesmall intestine without absorption, transit to the large intestine wherethe sugars can be decoupled to release CBD locally, avoiding systemicabsorption and delivery of the CBD to other tissues where it can haveunwanted effects.

Example 4 In Vitro Activity of Cannabidiol and TetrahydrocannabinolAgainst a Diverse Array of Microbes Including Aerobic and AnaerobicBacteria, Yeast, and Fungi INTRODUCTION

The antimicrobial activity of tetrahydrocannabinol (THC) and cannabidiol(CBD) was evaluated. The following example describes determining theMinimal Inhibitory Concentration (μg/mL) of these two compounds usingindustry-accepted protocols published by the Clinical LaboratoryStandards Institute (CLSI; 1-6). In order to evaluate the spectrum ofantimicrobial activity, these compounds were tested against aerobicbacteria, anaerobic bacteria, yeast, and fungi. Each compound was testedalone and in combination as described below.

METHODS Test Compounds and Comparators

The tetrahydrocannabinol (THC) and cannabidiol (CBD) were stored at −20°C. prior to testing. The compounds were supplied in methanol at 10 mg/mL(CBD) or 1 mg/mL (THC). Stock solutions of each compound were preparedby drying the methanol suspension and resuspending in DMSO to provide an80× stock. The comparator drugs ciprofloxacin, metronidazole, andamphotericin B were provided by Micromyx. Stock solutions of thecomparators were prepared at 40× on the day of testing and allowed tostand for at least 1 hr prior to use to auto-sterilize. Solvent, andworking stock concentrations were as follows:

Test/Control Testing Range Agents Solvent/Diluent (μg/mL) THC—delta 9 -DMSO 128-0.002 THC CBD—Cannabidiol DMSO 128-0.002 Ciprofloxacin Water 64-0.002 Metronidazole DMSO 32-0.03 Amphotericin B DMSO 64-0.06

Test Organisms

The test organisms for the assay were clinical isolates from theMicromyx repository or reference strains acquired from the American TypeCulture Collection (ATCC, Manassas, Va.). Upon receipt, the isolateswere streaked onto the appropriate agar plates and incubated at optimalconditions for growth. Colonies were harvested from these plates and acell suspension was prepared in the appropriate media containingcryoprotectant. Aliquots were then frozen at −80° C. Prior to the assay,the isolates were streaked for isolation from frozen stocks onto theappropriate agar plates and incubated at optimal conditions for growth.

Test Media

The media employed for testing the isolates was Mueller Hinton Broth(MHB II; BD; Lot No. 5257869) for aerobic organisms. For streptococci,this medium was supplemented with 3% laked horse blood (LHB; ClevelandScientific, Bath, Ohio; Lot No. 369595). MIC testing of anaerobicbacteria was performed with Brucella broth (BD; Lot No. 6155858)supplemented with hemin (Sigma, St. Louis, Mo.; Lot No. SLB46854),vitamin K1 (Sigma; Lot No. MKBN5958V) and 5% LHB. Yeast and fungi weretested in RPMI 1640 (Hyclone Laboratories, Logan, Utah; Lot No.AZC184041A) buffered with MOPS from Calbiochem (Billerica, Mass.; LotNo. 2875157).

All the above media was prepared/stored according to guidelines from theClinical and Laboratory Standards Institute (CLSI;1-5).

Broth Microdilution MIC Testing

MIC values were determined using the broth microdilution method asrecommended by CLSI (1-5). Automated liquid handlers (Multidrop 384,Labsystems, Helsinki, Finland; Biomek 2000 and Biomek FX, BeckmanCoulter, Fullerton Calif.) were used to conduct serial dilutions andliquid transfers.

To prepare the drug mother plates, which would provide the serial drugdilutions for the replicate daughter plates, the wells of a standard96-well microdilution plate (Costar 3795, Corning Inc., Corning, N.Y.)were filled with 150 μL of the designated diluent in columns 2-12. Thewells of column 1 were filled with either 300 μL of 40× of theappropriate compound or 150 μL of either 40× or 80× of the test articlesto make final concentrations of the combinations as shown below. A 150μL volume of solution was transferred from the wells in Column 1 to maketen subsequent 2-fold serial dilutions from columns 2-11. The wells ofColumn 12 contained no drug and ultimately served as the organism growthcontrol wells. For the test articles, this resulted in the followingdilutions and concentrations in each row of the respective motherplates:

Mother Plate 1 Row No. → A B C D E F G H Dilution → 128- 2- 128/64- 2/1-128/128- 2/2- 64/128- 1/2- 0.12 0.002 0.12/0.06 0.002/0.001 0.12/0.120.002/0.002 0.06/0.12 0.001/0.002 THC → 40X 40X 80X 80X 80X 80X 40X 40XCBD → None None 40X 40X 80X 80X 80X 80X Ratio 1:0 1:0 2:1 2:1 1:1 1:11:2 1:2 THC:CBD Mother Plate 2 Row No. → A B Dilution → 128-0.12 2-0.002THC → None None CBD → 40X 40X Ratio 0:1 0:1 THC:CBD

The daughter plates were loaded with 185 μL per well of the appropriatetest medium for the tested organism. They were then completed with atransfer of 5 μL of drug solution from each well of the mother plate tothe corresponding well of each daughter plate in a single step. Daughterplates used for testing of anaerobes were allowed to pre-reduce inanaerobe boxes for at least 2 hr prior to inoculation.

A standardized inoculum of each organism was prepared per CLSI methods(1-5). Suspensions were prepared in sterile saline to equal theturbidity of a 0.5 McFarland standard, diluted in appropriate media perCLSI recommendations, and transferred to compartments of sterilereservoirs divided by width (Beckman Coulter). The Biomek 2000 (BeckmanCoulter, Fullerton Calif.) was used to inoculate all. Daughter platesfor the organisms were placed on the Biomek 2000 in reverse orientation.The Biomek 2000 delivered 10 μL of standardized inoculum into each wellof the appropriate plate resulting in a final concentration ofapproximately 5×10⁵ CFU/mL (bacteria), 0.5 to 2.5×10³ CFU/mL (yeast),and 0.2 to 2.5×10⁴ CFU/mL (filamentous fungi) per well.

Plates were stacked 3-4 high, covered with a lid on the top plate andincubated according to CLSI methodology. Aerobes were incubated forapproximately 16-20 hr, Streptococcus for 20-24 hr, and anaerobes,yeast, and fungi for 24-48 hr. After incubation, plates were viewed fromthe bottom using a plate viewer.

Un-inoculated solubility control plates for each test media and drugwere incubated in parallel with test plates and observed for evidence ofdrug precipitation. MICs were read where visible growth of the organismwas inhibited.

RESULTS AND DISCUSSION

Compound precipitation observed with the uninoculated solubilitycontrols is summarized in the table below.

Compound Precipitation Observed with the Uninoculated SolubilityControls

Compound and lowest concentration (μg/mL) at which precipitation wasobserved THC/CBD THC/CBD THC/CBD Media type THC CBD 2:1 1:1 1:2 MHB IIDCP none none DCP DCP 64, 128 128/128 64/128 64/64 32/64 MHB II withnone none none none none Blood Brucella Broth DCP 128 none none nonenone RPMI TRP none none TRP TRP 128, 64, 128/128, 64/128, 32, 16 64/64,32/32, 32/64, 16/16 16/32 None = (no precipitation observed); TRP =trace precipitate; DCP = distinct precipitate

Distinct or trace precipitation at the highest test concentration (128μg/mL) was observed for THC, THC/CBD 1:1, and THC/CBD 1:2 in MHB IIwithout blood, RPMI, and Brucella broth. However, the nature of theprecipitate permitted reading growth of the organisms through 128 μg/mL.

The activity of CBD, THC, and the control agents are shown in the tablesbelow. Results for the control agents (ciprofloxacin, metronidazole, andamphotericin B) were all within the established CLSI QC ranges (2, 6)for the respective agents and quality control organisms.

Against Gram-positive and -negative aerobic bacteria, CBD and THC testedalone were active against Gram-positive aerobes with MIC values of 1-4μg/mL, but were inactive against Gram-negative aerobes. The MIC valuesfor CBD were similar to those observed in a previous study.Ciprofloxacin activity was as expected for a broad spectrumanti-bacterial agent, with resistance apparent for the evaluated MRSA,VRE, and KPC-2 isolates where fluoroquinolone-resistance is commonlyencountered.

As shown in the table below, THC and CBD displayed similar activityagainst the evaluated anaerobes, with MICs from 16−>128 μg/mL (THC) and8−>128 μg/mL (CBD). MIC values for CBD were within 2-fold of a previousstudy, with the exceptions of B. bifidum where the previous MIC was 2μg/mL and B. fragilis where the previous MIC was 32 μg/mL. Metronidazolewas active against all organisms with the exception of the evaluatedLactobacillus spp. which is consistent with the anticipated activity forthese isolates.

Combinations of THC and CBD did not display enhanced anti-microbialactivity against evaluated microbes. This suggests that CBD and THC arefunctional analogs, and the antimicrobial activity is based on theirhydrophobicity and affinity for the cell membranes.

Neither THC nor CBD were active against the evaluated yeast and fungalisolates. Amphotericin B had the expected activity against both testisolates. In summary, CBD and THC were active against Gram-positiveaerobic bacteria (S. aureus, E. faecalis, S. pneumoniae, and S.pyogenes) and both obligate and facultative anaerobes (C. difficile,Bifidobacterium spp., and Lactobacillus spp.), but not Gram-negativeaerobic bacteria, yeast, and fungi.

Activity of THC, CBD, and THC:CBD Combinations Against Aerobic Bacteria

MIC (μg/mL) THC/CBD THC/CBD THC/CBD Cipro- Organism Isolate No. THC CBD2:1 1:1 1:2 floxacin Gram- Staphylococcus MMX 100 2 2 4/2 2/2 1/2 0.25positive aureus (MSSA) (ATCC 29213) (0.12-0.5)¹ Staphylococcus MMX 757 22 4/2 2/2 1/2 >64 aureus (MRSA) Enterococcus MMX 101 2 2 4/2 2/2 1/2 1faecalis (VSE) (ATCC 29212) (0.25-2)¹   Enterococcus MMX 848 2 2 4/2 2/21/2 64 faecalis (VRE) Streptococcus MMX 1195 1 4 8/4 1/1 1/2 0.5pneumoniae (ATCC 49619) Streptococcus MMX 404 16 32 64/32 64/64 32/640.25 pyogenes Gram- Escherichia MMX102 >128 >128 >128/64  >128/128  >64/128 0.008 negative coli (ATCC25922)  (0.004-0.015)¹ Klebsiella MMX4683 >128 >128 >128/64  >128/128  >64/128 >64 pneumoniae (KPC-2)Pseudomonas MMX 1380 >128 >128 >128/64  >128/128  >64/128 4 aeruginosaAcinetobacter MMX 1630 >128 >128 >128/64  >128/128  >64/128 4 baumanniiMSSA = methicillin-susceptible S. aureus; MRSA = methicillin-resistantS. aureus; VSE = vancomycin-susceptible enterococci; VRE =vancomycin-resistant enterococci; KPC-2 = Klebsiella pneumoniaecarbapenemase-positive ¹CLSI QC range shown in parenthesis

Activity of THC, CBD, and THC:CBD Combinations Against AnaerobicBacteria

MIC (μg/mL) THC/CBD THC/CBD THC/CBD Metroni- Organism Isolate No. THCCBD 2:1 1:1 1:2 dazole Clostridium MMX 4381 32 64 >128/64 64/64 32/640.25 difficile (ATCC 700057) Clostridium MMX 8330 32 64 >128/64 32/3232/64 0.25 difficile Clostridium MMX 8333 32 64  64/32 32/32 16/32 2difficile Lactobacillus MMX 4147 No No No No No No crispatus growthgrowth growth growth growth growth Lactobacillus MMX 4149128 >128 >128/64 >128/128  >64/128 >32 jensenii Bifidobacterium MMX 396516 8  8/4 4/4  8/16 2 bifidum Bifidobacterium MMX 3968 32 64 >128/6464/64 32/64 8 longum Bacteroides MMX123 >128 >128 >128/64 >128/128  >64/128 0.5 fragilis (ATCC 25285)(0.25-2)¹ ¹CLSI QC range shown in parenthesis

Activity of THC, CBD, and THC:CBD Combinations Against Fungi

MIC (μg/mL) at 24, 48 hr THC/CBD THC/CBD THC/CBD Amphoter- OrganismIsolate No. THC CBD 2:1 1:1 1:2 icin B Candida MMX 2323 >128(4¹), >128, >128/64 >128/128 >64/128 0.12 parapsilosis (ATCC 22019) >128(4¹) >128 (16/8¹), (32/32¹), (16/32¹),(0.5-2)³, >128/64 >128/128 >64/128 0.5  (16/8¹) (32/32¹) (16/32¹)(0.25-4)³  Aspergillus MMX5280 >128, >128 >128, >128/64, >128/128, >64/128, 0.25, 1 fumigatus(MYA-3626) >128 <128/64 >128/128 >64/128 (0.5-4)³  ¹50% inhibitionobserved at XXX μg/mL relative to the growth control ²50% inhibitionobserved at XXX μg/mL relative to the growth control ³CLSI QC rangesshown in parenthesis

Example 5 Determination of Minimal Inhibitory Concentration (μg/mL) andMinimum Bactericidal Concentration (MBC) of Cannabidiol andTetrahydrocannabinol for a Variety of Aerobic and Anaerobic Bacteria

The test agents (THC and CBD) were be dried and resuspended in 100% DMSOas described above. MIC/MBC testing utilized a drug concentration rangeof 128-0.12 μg/m L. For aerobic organisms only, imipenem was testedusing a concentration range of 8-0.008 μg/mL to serve as a qualitycontrol drug for MIC testing (MBC testing for imipenem was conducted forE. coli ATCC 25922 as a quality control MBC test). For anaerobictesting, metronidazole was tested as the quality control antibiotic forMIC testing using a concentration range of 32-0.03 μg/mL (no MBCtesting). In all 96-well plates, the twelfth well of each row did notcontain drug and will serve as the positive growth control.

The broth microdilution MIC methodology followed the proceduresdescribed by the Clinical Laboratory Standards Institute (CLSI) andemployed automated liquid handlers to conduct serial dilutions andliquid transfers. Following incubation, the microplates were removedfrom the incubator and viewed from the bottom using a Scienceware platereader. The solubility control plate was observed for evidence of drugprecipitation. The MIC was read and recorded as the lowest concentrationof drug that inhibited visible growth of the organism.

The MBC was evaluated for THC and CBD only, in parallel with the MIC inaccordance with CLSI guidelines. After determining the MIC,concentrations or wells at the MIC and up to 3 dilutions above the MICwere be sampled and plated to determine viable bacteria. Ten microliteraliquots were spotted in duplicate for each concentration/well sampled(the MIC and three wells above the MIC). The MBC was determined based ona pre-determined threshold which indicates a 99.9% decrease in viablebacteria post-incubation relative to the inoculum.

RESULTS AND DISCUSSION

As detailed in the table below CBD and THC exhibit antimicrobialactivity and are overall biocidal against the strains tested.

THC CBD Organism MIC MBC MBC:MIC³ MIC MBC MBC:MIC S. aureus 4 4 1 1 2 2ATCC 29213 S. aureus 4 32 8 1 1 1 MMX 757 E. faecalis 2 8 4 2 2 1 ATCC29212 E. faecalis 2 4 2 1 2 2 MMX 848 S. pneumoniae 1 ID NA 1 4 4 ATCC49619 S. pyogenes 32 32 1 32 32 1 MMX 404 E. coli >256 NT NA >256 NT NAATCC 25922 C. difficile 64 128 2 16 32 2 ATCC 700057 C. difficile 64 641 32 32 1 MMX 8330 C. difficile 64 128 2 16 32 2 MMX 8333 B. bifidum 32128 4 16 32 2 MMX 3965 B. longum 32 >256 >8  16 64 4 MMX 3968 B.fragilis >256 NT NA 16 NT NA ATCC 25285 ID: indeterminate, NT: nottested, NA: not applicable 1 CLSI QC range shown in parenthesis ³MBC:MICratios of 1-4 are indicative of bactericidal activity.

Control Agents

Imipenem Metronidazole Organism MIC MBC MBC:MIC MIC MBC MBC:MIC S.aureus 0.015 NA NA NT NA NA ATCC 29213 (0.015-0.06)¹  S. aureus 4 NA NANT NA NA MMX 757 E. faecalis 0.5 NA NA NT NA NA ATCC 29212 (0.5-2)¹   E.faecalis 1 NA NA NT NA NA MMX 848 S. pneumoniae 0.015 NA NA NT NA NAATCC 49619 (0.03-0.12)¹ S. pyogenes ≤0.008 NA NA NT NA NA MMX 404 E.coli 0.12 0.12 1 NT NA NA ATCC 25922 (0.06-0.25)¹ (0.12-0.5)¹ C.difficile NT NA NA 0.12 NA NA ATCC 700057 C. difficile NT NA NA 0.5 NANA MMX 8330 C. difficile NT NA NA 0.5 NA NA MMX 8333 B. bifidum NT NA NA1 NA NA MMX 3965 B. longum NT NA NA 8 NA NA MMX 3968 B. fragilis NT NANA 0.5 NA NA ATCC 25285 (0.25-2)¹ ID: indeterminate, NT: not tested, NA:not applicable ¹CLSI QC range shown in parenthesis 3 MBC:MIC ratios of1-4 are indicative of bactericidal activity.Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims. Furthermore, the term “comprising of” includesthe terms “consisting of” and “consisting essentially of.”

REFERENCES

-   1.) Clinical Laboratory and Standards Institute. Methods for    Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow    Aerobically; Approved Standard—Ninth Edition. CLSI document M07-A10    [ISBN 1-56238-783-9]. CLSI, 950 West Valley Road, Suite 2500, Wayne,    Pa. 19087 USA, 2015.-   2.) CLSI. Performance Standards for Antimicrobial Susceptibility    Testing; Twenty-Seventh Informational Supplement. CLSI document    M100-S27. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pa.    19087-1898 USA, 2017.-   3.) CLSI. Methods for Antimicrobial Susceptibility Testing of    Anaerobic Bacteria; Approved Standard—Seventh Edition. CLSI document    M11-A7 (ISBN 1-56238-626-3). CLSI, 940 West Valley Road, Suite 1400,    Wayne, Pa. 19087-1898 USA, 2007.-   4.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Yeasts; Approved Standard—Third Edition.    CLSI document M27-A3 [ISBN 1-56238-666-2]. CLSI, 940 West Valley    Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2008.-   5.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Filamentous Fungi; Approved    Standard—Second Edition. CLSI document M38-A2 [ISBN 1-56238-668-9].    CLSI, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA,    2008.-   6.) CLSI. Reference Method for Broth Dilution Antifungal    Susceptibility Testing of Yeasts; Fourth Informational Supplement.    CLSI document M27-S4. CLSI, 940 West Valley Road, Suite 1400, Wayne,    Pa. 19087-1898 USA, 2012.-   7.) Ali, E. M. M., et al. (2012). Antimicrobial Activity of Cannabis    sativa L. Chinese Medicine. 3:61-64.-   8.) Appendino, G., et al. (2008). Antibacterial Cannabinoids from    Cannabis sativa: A Structure-Activity Study. J Nat Prod. 71(8):    1427-1430.-   9.) CDC (2013). Antibiotic/Antimicrobial Resistance: Biggest    Threats. Accessed on 2017 May 8:    https://www.cdc.gov/drugresistance/biggest_threats.html-   10.) CDC. (2015). Nearly half a million Americans suffered from    Clostridium difficile infections in a single year. Press release.    Accessed on 2017 May 8:    https://www.cdc.gov/media/releases/2015/p0225-clostridium-difficile.html-   11.) Gigli, S., et al. (2017). Cannabidiol restores intestinal    barrier dysfunction and inhibits the apoptotic process induced by    Clostridium difficile toxin A in Caco-2 cells. United European    Gastroenterology J. 0(0): 1-8.-   12.) Kabelik, J. et al. (1960). Cannabis as a medicament. UN Office    on Drugs and Crime. Accessed on 20170508:    https://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1960-01-01_3_page003.html-   13.) van Klingeren, B., Ten Ham, M. (1976). Antibacterial activity    of (delta9)-tetrahydrocannabinol and cannabidiol. Antonie van    Leeuwenhoek. 42(1976): 9-12.-   14.) WHO. (2014). Antimicrobial Resistance, Global Report on    Surveillance.-   15.) WHO. (2017). Global priority list of antibiotic-resistant    bacteria to guide research, discovery, and development of new    antibiotics. Accessed on 2017 May 8:    http://www.who.int/medicines/publications/WHO·PPL-Short_Summary_25Feb-ET_NM_WHO.pdf-   16.) CLSI. Methods for Determining Bactericidal Activity of    Antimicrobial Agents; Approved Guideline. CLSI document M26-A. CLSI,    940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 1999.

1-15. (canceled)
 16. A method of treating an infection comprisingadministering to a subject in need thereof, an effective amount of atleast one cannabinoid compound, wherein the at least one cannabinoidcompound is cannabidiol, tetrahydrocannabinol, or combinations thereof,and wherein the infection is an E. faecalis infection, a S. pneumoniainfection, a C. difficile infection, a L. cripatus infection, a B.bifidum infection, a B. longum infection, or combinations thereof. 17.The method of claim 16, wherein the at least one cannabinoid compound iscannabidiol.
 18. The method of claim 17, wherein the infection is a C.difficile infection.
 19. The method of claim 18, wherein the effectiveamount of the cannabidiol is administered orally.
 20. The method ofclaim 17, wherein the infection is an E. faecalis infection.
 21. Themethod of claim 20, wherein the effective amount of the cannabidiol isadministered orally.
 22. The method of claim 16, wherein the at leastone cannabinoid compound is tetrahydrocannabinol.
 23. The method ofclaim 22, wherein the infection is a C. difficile infection.
 24. Themethod of claim 23, wherein the effective amount of thetetrahydrocannabinol is administered orally.
 25. The method of claim 23,wherein the infection is an E. faecalis infection.
 26. The method ofclaim 25, wherein the effective amount of the tetrahydrocannabinol isadministered orally.
 27. The method of claim 16, wherein the effectiveamount of the at least one cannabinoid compound is administered orally.28. The method of claim 16, wherein the effective amount of the at leastone cannabinoid compound is administered topically.
 29. A method oftreating microbial infections of the gastrointestinal tract in a subjectin need thereof, comprising administering an effective amount of atleast one cannabinoid compound, wherein the at least one cannabinoidcompound is cannabidiol, tetrahydrocannabinol, or combinations thereof,wherein the microbial infection is a C. difficile infection, an E.faecalis infection, or a combination thereof.
 30. The method of claim29, wherein the microbial infection is a C. difficile infection.
 31. Themethod of claim 30, wherein the at least one cannabinoid compound iscannabidiol.
 32. The method of claim 30, wherein the at least onecannabinoid compound is tetrahydrocannabinol.
 33. The method of claim29, wherein the microbial infection is an E. faecalis infection.
 34. Themethod of claim 33, wherein the at least one cannabinoid compound iscannabidiol.
 35. The method of claim 33, wherein the at least onecannabinoid compound is tetrahydrocannabinol.